VEGF-A121 assay

ABSTRACT

The invention provides a method for enriching the level of VEGF-A 121  isoform in a sample by selectively removing the VEGF-A 165  isoform from the sample using a neuropilin-1 pull-down procedure, then determining the total amount of VEGF-A remaining afterward. The invention provides methods of treating a patient suffering from a disease which may benefit from the administration of a VEGF antagonist by determining the level or ratio of VEGF-A 121  in the patient&#39;s circulation. Methods of diagnosis, prognosis, monitoring, and patient stratification are also provided.

STATEMENT OF RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/900,129, filed on May 22, 2013, now allowed, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.61/649,959, filed 22 May 2012, all of which applications are hereinspecifically incorporated by reference.

FIELD OF THE INVENTION

The invention relates to assays for measuring a VEGF-A₁₂₁ isoform. Theinvention also relates to methods for treating patients with VEGFrelated diseases by predicting whether a patient is likely to respond toVEGF antagonist therapy by assessing the level of VEGF-A₁₂₁ in thepatient's blood.

BACKGROUND OF THE INVENTION

Cancer is a collection of myriad diseases, each of which has a uniquemolecular signature and drug response profile. For example, colorectalcancer may be any one of hereditary nonpolyposis colorectal cancer,familial adenomatous polyposis, and Gardner syndrome. Furthermore, anyparticular cancer may be associated with distinct molecular lesions,such as wnt signaling dysregulation, p53 and other DNA repair andapoptosis dysregulation, growth factor signaling and other oncogenedysregulation, or any combination thereof. Thus, cancer, such asprostate cancer, breast cancer, colorectal cancer, lung cancer, orpancreatic cancer, may be further subdivided into specific moleculardiseases, each of which has its own diagnostic or prognostic indicatorsand therapeutic responses.

There is a continuing medical need for specific tests to diagnose theparticular nature of a cancer, and to determine whether a patient islikely to respond to a particular therapy. VEGF antagonists are a classof therapeutics intended to treat cancer by abrogating or otherwisealtering the vascularization of tumors. The effectiveness of these drugsdepends in part on the specific type of cancer and other factorsassociated with the physiology of the patient. The development of aprognostic biomarker assay or patient stratification protocol willenable the physician to select those patients who are most likely torespond favorably to therapy, and to monitor a patient's response to atherapy.

Several biomarkers can predict how a patient will respond to VEGFantagonist (e.g., aflibercept and bevacizumab) therapy. Those biomarkersinclude E-selectin, bFGF, ICAM, and VEGF (Calleri et al., Clin CancerRes 2009, 15(24):7652-7657; Dowlati et al., Clin Cancer Res 2008,14(5):1407-1412.) VEGF is found in several isoforms: VEGF-A₂₀₆,VEGF-A₁₈₉, VEGF-A₁₆₅, VEGF-A₁₄₅, VEGF-A₁₂₁, VEGF-B, VEGF-C and VEGF-D.VEGF-A₂₀₆, VEGF-A₁₈₉ and VEGF-A₁₆₅, bind to neuropilin-1 (“NRP1”) aswell as VEGF receptors 1 and 2 (reviewed in Neufeld et al., FASEB J.,1999, 13:9-22).

SUMMARY OF THE INVENTION

VEGF-A₁₆₅ and VEGF-A₁₂₁ are the predominant forms found in thecirculation. VEGF-A₁₆₅ is subject to high patient-to-patient variabilityand is rapidly cleared from the circulation; and therefore its levelsare not an accurate prognostic or predictive biomarker for VEGFantagonist therapy. VEGF-A₁₂₁ on the other hand is expected to be a moreaccurate biomarker for VEGF antagonist therapy.

The inventors have made the surprising discovery that levels ofVEGF-A₁₂₁ can be more accurately determined in a sample by removingVEGF-A₁₆₅ from the sample using a NRP1 pull-down procedure.

Thus, in one aspect, the invention provides a method for determining thelevel of VEGF-A₁₂₁ in a sample, by combining the sample with NRP1, whichforms a complex with the VEGF-A₁₆₅; and subsequently removing thecomplex from the sample, leaving behind a sample depleted of VEGF-A₁₆₅and enriched for VEGF-A₁₂₁. The VEGF-A₁₆₅-depleted sample is thensubjected to a VEGF-A quantitative assay, such as ELISA using a VEGF-Aantibody, which measures the level of VEGF-A₁₂₁ remaining in the sample.In one embodiment, the NRP1 is fixed to a substrate (e.g., a well orbead), and the sample is subsequently added to the substrate. After theNRP1-VEGF-A₁₆₅ complex forms, the sample is decanted and subjected tothe VEGF-A quantitative assay.

In another aspect, the invention provides a method of treating a patientsuffering from a VEGF-mediate disease, including cancer or ischemia.Prior to the administration of the VEGF antagonist, the levels ofVEGF-A₁₂₁ in the sample are determined by employing the NRP1 pull-downassay described above. The sample may be blood, serum, plasma, or thelike.

In yet another aspect, the invention provides a method of selectingpatient to be treated with a VEGF antagonist (i.e., patientstratification), a subset of which is the monitoring of a patient'sresponse to VEGF antagonist therapy and reassessing their responsivenessto the therapy. The method comprises the step of determining the levelof VEGF-A₁₂₁ in a patient, and then placing the patient either in acategory of a patient who is likely to respond to VEGF antagonisttreatment (category A), or in a category of a patient who is not likelyto respond to VEGF antagonist treatment (category B). When the level ofVEGF-A₁₂₁ is at or above a certain threshold for a particular patient,then that patient is in category A. When the level of VEGF-A₁₂₁ is belowa certain threshold for a particular patient, then that patient is incategory B. In some embodiments, the threshold level of VEGF-A₁₂₁ isabout 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to85, or 85 to 90 ρg/mL.

In another aspect, the invention provides a method of selecting apatient capable of responding to VEGF antagonist therapy comprising thesteps of (a) obtaining a biological sample from a patient; (b)determining the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio in the sample; and (c)selecting a patient whose sample contains VEGF-A₁₂₁/VEGF-A₁₆₅ ratio in arange of about 0.1 to 1000, wherein said patient is more likely torespond favorably to VEGF antagonist therapy than a patient havingVEGF-A₁₂₁/VEGF-A₁₆₅ ratio outside of the range.

In another aspect, the invention provides a method for treating cancerin a subject, comprising: (a) determining a VEGF-A₁₂₁/VEGF-A₁₆₅ ratio ina biological sample from said subject; and (b) based on the results of(a), administering a VEGF antagonist to the subject in need oftreatment. The method comprises determining the VEGF-A₁₂₁/VEGF-A₁₆₅ratio comprising (a) contacting a sample with a neuropilin 1 (“NRP1”)protein, wherein the VEGF-A₁₆₅ binds to the NRP1 protein to form aNRP1-VEGF-A₁₆₅ complex; (b) separating the VEGF-A₁₂₁ from theNRP1-VEGF-A₁₆₅ complex to produce a VEGF-A₁₆₅-depleted fraction; (d)quantifying the VEGF-A₁₂₁ in the VEGF-A₁₆₅-depleted fraction; (e)quantifying the VEGF-A₁₆₅ in the NRP1-VEGF-A165 complex; and (f)determining the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio based on the quantities in (d)and (e). In some embodiments, the ratio is about 0.001 to 1000, or about0.01 to 1000, or about 0.1 to 100, or about 0.1 to 10, or about 1 to 10.

The patient may suffer from any one or more of an angiogenesis orVEGF-dependent disease, such as metastatic breast cancer, gastriccancer, pancreatic cancer, renal cell carcinoma, non-small cell lungcancer, androgen-independent prostate cancer, ovarian cancer,adenocarcinoma, and inter alia colorectal cancer.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused in this specification is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention is defined by the claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure.

Unless defined otherwise, all technical and scientific terms used inthis application have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described in thisspecification can be used in the practice of the present invention,particular methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference in their entirety.

VEGF-A121 Assay

It is generally known that VEGF-A can serve as a predictive andprognostic biomarker for VEGF antagonist therapy; and that thepredominant isoforms of VEGF in the blood are VEGF-A₁₆₅ and VEGF-A₁₂₁.It is also known that VEGF-A₁₆₅ levels show significantpatient-to-patient variability, which makes it less than desirable as abiomarker. The quantification of VEGF-A₁₂₁ thus serves as a betterpredictive or prognostic biomarker, versus VEGF-A₁₆₅ or a mixture of the165 and 121 isoforms.

Applicants have discovered that by adding to a sample NRP1, which formsa complex with VEGF-A₁₆₅, but not VEGF-A₁₂₁, and pulling down theresultant NRP1-VEGF-A₁₆₅ complex, the remaining supernatant was depletedof VEGF-A₁₆₅. The remaining VEGF-A in the sample was thus relativelyenriched for VEGF-A₁₂₁. The total VEGF-A was then measured by aquantitative assay, such as via an anti-VEGF ELISA, and the total amountof VEGF-A₁₂₁ calculated from the total remaining VEGF-A.

Thus, in one aspect, the invention provides a method for determining theamount of VEGF-A₁₂₁ in a sample comprising the steps of (a) contacting asample comprising VEGF-A₁₆₅ and VEGF-A₁₂₁ with a NRP1 protein, whereinthe VEGF-A₁₆₅ binds to the NRP1 to form a NRP1-VEGF-A₁₆₅ complex; (b)separating the VEGF-A₁₂₁ from the NRP1-VEGF-A₁₆₅ complex to form aVEGF-A₁₆₅-depleted fraction; and (c) quantifying the VEGF-A₁₂₁ in theVEGF-A₁₆₅-depleted fraction.

The NRP1 may be fixed to a solid support. In one embodiment, the NRP1protein is fixed to the surface of a plate or a well within a microtiterplate. In another embodiment, the NRP1 is fixed to the surface of abead. Bead technology is well-known in the art. To affix the NRP1protein to the bead or well or plate, the bead or well or plate isincubated with a 1 μg/mL to 10 μg/mL NRP1 protein solution. In oneembodiment, the NRP1 protein solution is 10 μg/m L.

In one embodiment, NRP1 protein is covalently coupled to the surface ofbeads, (i.e. microspheres) such as carboxylated polystyrene microspheresby Luminex Corporation (Luminex Corporation, Austin, Tex., USA). In someembodiments, the beads are magnetic and/or emit fluorescence.Microsphere or bead kits, such as Luminex kits, are commerciallyavailable. Covalent coupling of the protein, capture, and separation ofthe microspheres is performed following the procedures recommended bythe manufacturer. In some embodiments, an NRP1-bead pull-down method todeplete VEGF-A₁₆₅ from the sample is used in accordance with theinvention described herein.

In some embodiments, the NRP1 protein is a human NRP1 protein or afraction thereof. In some embodiments, the NRP1 protein is a fusionprotein comprising of an Fc domain fused to a NRP1 domain. It is knownin the art that NRP1 binds to VEGF-A₁₆₅ via its B1B2 domain, which iswithin the extracellular domain (ecto-domain) of NRP1 (Mamluk et al.,2002, J. Biol. Chem., 277(27):24818-24825; Nakamura et al., 1998,Neuron, 21:1083-1100). Therefore, in some embodiments, the NRP1 proteincomprises the B1B2 domain. In some embodiments, the NRP1 proteincomprises the ecto-domain of NRP1, which itself contains the B1B2domain. In some embodiments, the ecto-domain or the B1B2 domain may befused to an Fc domain, for example a mouse Fc domain or a human Fcdomain (Dumont et al., 2006, Drug Development, 20(3):151-160). In oneembodiment, the NRP1 protein is a NRP1-B1B2-Fc fusion protein. Inanother embodiment, the NRP1 protein is a NRP1-ectodomain-Fc fusionprotein. NRP1 proteins are generally described in Hamerlik et al., J.Exp. Med. 2012 209(3):507-520; and Finley & Popel, AAPS J. 1 May 2012electronic publication.

In some embodiments, prior to the pull-down step, heparin may be addedto the VEGF-A-NRP1 protein admixture, which may enhance the formation ofa ternary complex containing VEGF-A₁₆₅. An additional 10% or moredepletion of VEGF-A₁₆₅ may be accomplished by the addition of heparin.The heparin may be at a concentration of from about 1 μg/mL to about 10μg/mL.

The NRP1 pull-down method described herein is capable of depleting up to75% of the VEGF-A₁₆₅. In some embodiments, at least 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%. 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or at least 75% of the VEGF-A₁₆₅is depleted from the sample. After the VEGF-A₁₆₅ is depleted, theremaining VEGF-A, which is predominantly VEGF-A₁₂₁, is quantified. Insome embodiments, the remaining VEGF-A is quantified via an antibodyassay, such as ELISA. However, one of ordinary skill in the art in thepractice of the invention may apply any method to quantify the remainingVEGF-A.

In some embodiments, the sample is a biological sample obtained from apatient. The sample may be a blood sample, or a derivative of blood,such as plasma or serum.

Methods of Treating Cancer

In one aspect, the invention provides a method of treating cancer in apatient, in which a step in the treatment regimen includes thedetermination of the concentration of VEGF-A₁₂₁ in the blood of thepatient. The cancer may be a cancer that is likely to respond to VEGFantagonist treatment, and includes but is not limited to such cancers asmetastatic breast cancer, gastric cancer, pancreatic cancer, renal cellcarcinoma, non-small cell lung cancer, androgen-independent prostatecancer, metastatic androgen-independent prostate cancer, ovarian cancer,adenocarcinoma, metastatic colorectal cancer, and inter alia colorectalcancer.

In one embodiment, the method of treating cancer includes the steps of(a) obtaining a sample from a patient who suffers from cancer; (b)determining the concentration of VEGF-A₁₂₁ in the sample; and (c)administering a VEGF antagonist. The sample may be a blood sample takenfrom the patient, which may be subsequently separated into plasma orserum, or otherwise manipulated prior to assessing the relative levelsof VEGF-A₁₂₁.

In another embodiment, the method for treating cancer in a subject,comprises: (a) determining a VEGF-A₁₂₁/VEGF-A₁₆₅ ratio in a biologicalsample from said subject; and (b) based on the results of (a),administering a VEGF antagonist to the subject in need of treatment.

In other embodiments, the method for treating cancer comprisesdetermining the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio comprising (a) contacting asample with a neuropilin 1 (“NRP1”) protein, wherein the VEGF-A₁₆₅ bindsto the NRP1 protein to form a NRP1-VEGF-A₁₆₅ complex; (b) separating theVEGF-A₁₂₁ from the NRP1-VEGF-A₁₆₅ complex to produce aVEGF-A₁₆₅-depleted fraction; (d) quantifying the VEGF-A₁₂₁ in theVEGF-A₁₆₅-depleted fraction; (e) quantifying the VEGF-A₁₆₅ in theNRP1-VEGF-A₁₆₅ complex; and (f) determining the VEGF-A₁₂₁/VEGF-A₁₆₅ratio based on the quantities in (d) and (e).

In some embodiments, the ratio is about 0.001 to 1000, or about 0.01 to1000, or about 0.1 to 100, or about 0.1 to 10, or about 1 to 10. In someembodiments, the ratio is about 0.1 to about 1000. In other embodiments,the ratio is greater than about 0.1, or greater than about 10, orgreater than about 100.

In still other embodiments, the biological sample is blood, plasma orserum.

In some embodiments, the VEGF-antagonist is a biotherapeutic molecule,such as a “trap”, an antibody, or a fragment thereof. For example, theVEGF-antagonist may be a human or humanized monoclonal antibody thatblocks VEGF activity. In some embodiments, the VEGF antagonist may beany one or more of bevacizumab, ranibizumab, aflibercept, andramucirumab; alone or in combination with one or more other drugs.Bevacizumab is an anti-VEGF monoclonal antibody, which inhibitsangiogenesis and is considered to be an anti-cancer drug (Los et al.,2007, The Oncologist 12(4): 443-50). Ranibizumab is a Fab fragment ofthe bevacizumab antibody and is used as an anti-angogenesis therapeutic(Martin et al., 2011, New England Journal of Medicine364(20):1897-1908). Aflibercept is a “VEGF-Trap” molecule, comprisingthe extracelluar domains of VEGFR-1 and VEGFR-2, and the Fc region ofimmunoglobulin G (IgG) (Takahashi, 2011, Biol. Pharm. Bull.34(12):1785-1788). Aflibercept is an emerging first-line treatmentoption for androgen-independent prostate cancer and a second-linetherapy for metastatic colorectal cancer (George and Moul, 2012,Prostate 72(3):338-349). Ramucirumab is a human monoclonal antibodydirected against VEGFR-2, which functions as a receptor antagonist (Hsuand Wakelee, 2009, Biodrugs 23(5): 289-304).

In some embodiments, the amount of VEGF-A₁₂₁ in the patient sample isdetermined by contacting the sample with a NRP1 protein, wherein anyVEGF-A₁₆₅ in the sample binds to the NRP1 to form a NRP1-VEGF-A₁₆₅complex, which is subsequently separated from the remaining VEGF-A₁₂₁,which is then quantified using a standard VEGF assay.

The NRP1 protein, various embodiments of which are as described above,may be fixed to a solid support, such as e.g., beads or the surface of aplate or well. In one embodiment, the NRP1 protein is fixed to a plateor well of a microtiter plate. To fix the NRP1 protein to the well orplate, the well or plate is incubated with 1 μg/mL to 10 μg/mL NRP1protein. In one embodiment, the NRP1 protein is at 10 μg/mL.

In some embodiments, heparin may be added to the VEGF-A-NRP1 proteinadmixture, which may enhance the formation of a ternary complexcontaining VEGF-A₁₆₅. In some cases, the heparin enhances complexformation by at least 10%. The heparin may be at a concentration of fromabout 1 μg/mL to about 10 μg/mL.

The NRP1 pull-down method described herein is capable of depleting up to75% of the VEGF-A₁₆₅ in a sample. In some embodiments, at least 35%,36%, 37%, 38%, 39%, s40%, 41%, 42%. 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or at least 75% ofthe VEGF-A₁₆₅ is depleted from the sample. After the VEGF-A₁₆₅ isdepleted, the remaining VEGF-A, which predominantly comprises VEGF-A₁₂₁,is quantified. In some embodiments, the remaining VEGF-A is quantifiedvia an antibody assay, such as ELISA. However, one of ordinary skill inthe art may apply any method to quantify the remaining VEGF-A in thepractice of the invention.

Patient Stratification

In one aspect, the invention provides a method for selecting a patientcapable of responding to VEGF antagonist therapy comprising the steps ofobtaining a sample from a patient, determining the amount of VEGF-A₁₂₁in the sample, and determining whether the patient is likely to respondfavorably to VEGF antagonist therapy. Thus, VEGF-A₁₂₁ is used as abiomarker for the diagnosis of an VEGF-dependent cancer, for theprognosis of the development of the VEGF-dependent cancer, formonitoring patient response to VEGF antagonist therapy, or to stratifypatients into at least two groups: those who are more likely to respondto VEGF antagonist therapy, and those who are not.

In another aspect, the invention provides a method of selecting apatient capable of responding to VEGF antagonist therapy comprising thesteps of (a) obtaining a biological sample from a patient; (b)determining the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio in the sample; and (c)selecting a patient whose sample contains VEGF-A₁₂₁/VEGF-A₁₆₅ ratio in arange of about 0.1 to 1000, wherein said patient is more likely torespond favorably to VEGF antagonist therapy than a patient havingVEGF-A₁₂₁/VEGF-A₁₆₅ ratio outside of the range.

In some embodiments, the ratio is about 0.001 to 1000, or about 0.01 to1000, or about 0.1 to 100, or about 0.1 to 10, or about 1 to 10. Inother embodiments, the ratio is greater than about 0.1, or greater thanabout 10, or greater than about 100.

In still other embodiments, the biological sample is blood, plasma orserum

In some embodiments, the patient suffers from a cancer or otherangiogenesis-related disease, such as ischemia. Those cancers to whichthe invention is directed for example include but are not limited tometastatic breast cancer, gastric cancer, pancreatic cancer, renal cellcarcinoma, non-small cell lung cancer, androgen-independent prostatecancer, metastatic androgen-independent prostate cancer, ovarian cancer,adenocarcinoma, metastatic colorectal cancer and inter alia colorectalcancer.

In some embodiments, the VEGF antagonist therapy includes theadministration of a biotherapeutic molecule alone or in combination withanother cancer therapeutic (e.g., pacitaxel). Exemplary VEGF antagonistbiotherapeutics include bevacizumab, ranibizumab, aflibercept, andramucirumab, as described above.

In some embodiments, a favorable response to VEGF antagonist therapymeans the slowing of the rate of growth of cancer in the patient. Insome embodiments, a favorable response means the ability to enhance theeffects of another chemotherapy. In some embodiments, a favorableresponse is the extension of life.

For patient stratification, a patient is selected as a prospectiveresponder to VEGF antagonist therapy by determining whether the levelsof VEGF-A₁₂₁ in her circulation meets or exceeds a particular thresholdlevel. The level of the VEGF-A₁₂₁ biomarker is determined in a NRP1pull-down assay as described above and exemplified below.

The ratio of VEGF-A₁₂₁ to VEGF-A₁₆₅ in blood, plasma, or serum may alsobe used for patient stratification, diagnosis and prognosis, andpredicting whether a patient will respond to VEGF antagonist therapy.The expression level of the VEGF-A₁₂₁ biomarker and that of theVEGF-A₁₆₅ biomarker is determined in the NRP1 pull-down assay asdescribed herein, and the ratio is calculated based on the molar amountof each species in the patient sample. As used hereinafter, the term“VEGF-A₁₂₁/VEGF-A₁₆₅ ratio” refers to the number of moles of VEGF-A₁₂₁protein divided by the number of moles of VEGF-A₁₆₅ protein in thesample (plasma, serum, blood or the like). The level of VEGF-A₁₂₁ andVEGF-A₁₆₅ in a sample can be measured by any method known in the artdesigned to specifically measure the different VEGF-A isoforms. Incertain embodiments, the levels of VEGF-A₁₂₁ and VEGF-A₁₆₅ protein in abiological sample are determined by separately measuring the levels ofVEGF-A₁₂₁ and VEGF-A₁₆₅ protein using the NRP-1 pull-down assaydescribed herein.

In one embodiment, the VEGF-A₁₂₁ is quantified in the sample followingthe NRP1-VEGF-A₁₆₅ pull-down procedure. In another embodiment, theVEGF-A₁₆₅ is quantified from the NRP1-VEGF-A₁₆₅ complex separated fromthe sample. Other methods known by one of ordinary skill in the art maybe used to determine the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio in a biologicalsample, and the NRP-1 pull-down assay is one example.

In one embodiment, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is about 0.001 to about1000. In some embodiments, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is about 0.01to about 1000. In other embodiments, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio isabout 0.1 to about 100. In certain embodiments, the ratio is between therange of about 0.1 to about 10, or about 1 to about 10, or about 1 toabout 100. In some embodiments, the ratio is about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10.

In some embodiments, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is about 10 to about100. In some embodiments, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is about 100 toabout 200. In other embodiments, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is about200 to about 300, or about 300 to about 400, or about 400 to about 500,or about 500 to about 600, or about 600 to about 700, or about 700 toabout 800, or about 800 to about 900, or about 900 to about 1000.

In some embodiments, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is greater than about0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7,about 0.8, about 0.9, or greater than about 1. In some embodiments, theVEGF-A₁₂₁/VEGF-A₁₆₅ ratio is greater than about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, or greater thanabout 10. In some embodiments, the VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is greaterthan about 10, about 20, about 30, about 40, about 50, about 60, about70, about 80, about 90, or greater than about 100.

The inventors envision that the ratio of VEGF-A₁₂₁ to VEGF-A₁₆₅ in apatient sample (blood, serum, plasma, or the like) is useful forselecting a patient who will likely benefit from or respond to VEGFantagonist therapy. The VEGF-A₁₂₁/VEGF-A₁₆₅ ratio is also envisioned tobe a biomarker for the diagnosis of an VEGF-dependent cancer, for theprognosis of the development of the VEGF-dependent cancer, for theprognosis of the survival of the patient suffering from VEGF-dependentcancer, for monitoring patient response to VEGF antagonist therapy, orto stratify patients into at least two groups: those who are more likelyto respond to VEGF antagonist therapy, and those who are not.

Patients with increased pretreatment circulating levels of VEGF-A weremore sensitive to treatment and were shown to correlate with betterprognosis of cancer survival, such as increased progression-freesurvival and overall survival (Miles et al, D. et al., Jul. 10, 2010, JClin Oncology 28(20):3239-3247; Van Cutsem, et al. Jun. 10, 2012, J ClinOncology 30(17):2119-2127; Lambrechts et al., Mar. 20, 2013, J ClinOncology, 31(9):1219-1230). Direct protein quantification methodsselective for only VEGF-A₁₂₁ have not been identified. An ELISA assay,which may preferentially identify shorter VEGF forms, but alsoidentifies longer VEGF-A isoforms, has been used as a prognostic orpredictive biomarker (see International Publication Nos. WO2012010547,WO2012010548 and WO2012010550). A more selective method which identifiesand quantifies both VEGF-A₁₂₁ protein and VEGF-A₁₆₅ protein in abiological sample is therefore highly desirable. Without wishing to bebound by any one theory, a ratio of the two predominant forms ofcirculating VEGF-A protein, i.e. VEGF-A₁₂₁ and VEGF-A₁₆₅, is envisionedto be a more accurate biomarker (as a diagnostic, prognostic, andtreatment response biomarker) for patients afflicted with aVEGF-dependent cancer. The method described herein therefore provides asimple and robust assay for the measurement of such biomarkers.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Depletion Studies

VEGF-A₁₆₅ was diluted in PBS 0.5% BSA, and then transferred to an ELISAplate coated with either an NRP1-Fc fusion protein or BSA control. Somewells were coated with 1 μg/mL, and others with 10 μg/mL of a NRP1-Fcfusion protein. Two different NRP1-Fc fusion protein constructs wereemployed here: (1) NRP1-ecto-mFc, which comprises the ectodomain ofhuman NRP1 (see Nakamura et al., Neuron 1998, 21:1093-1100) fused tomurine Fc; and (2) NRP1-B1B2-mFc, which comprises the B1B2 domains ofhuman NRP1 (see Mamluk et al., J. Biol. Chem. 2002, 27(5):24818-24825)fused to murine Fc. 100, 200, or 400 ρg/mL of the VEGF-A₁₆₅ was appliedto the coated wells and incubated for four hours or overnight to allowfor binding between VEGF-A₁₆₅ and NRP1 to occur. The well supernatantswere removed and the levels of VEGF-A₁₆₅ remaining in those supernatantswere determined by ELISA.

The results are presented in Table 1. To summarize, as much as 67% and57% of the VEGF-A₁₆₅ was depleted in the wells coated in 10 μg/mL and 1μg/mL NRP1-ecto-mFc, respectively. In addition, 10 μg/mL NRP1-B1B2-mFcdepleted as much as 59% of the VEGF-A₁₆₅.

TABLE 1 Starting Average Concen- Remaining Remaining tration Concen-Concen- of VEGF₁₆₅ tration tration ± Depletion Coating (ρg/mL) Well(ρg/mL) SD of VEGF₁₆₅ NRP1- 400 A1 220 190 ± 42 53% ecto-mFc A2 160 (1μg/mL) 200 B1 90  90 ± 0.6 55% B2 89 100 C1 47 43 ± 6 57% C2 39 NRP1-400 A3 170 159 ± 15 60% ecto-mFc A4 148 (10 μg/mL) 200 B3 75 72 ± 4 64%B4 69 100 C3 35 34 ± 2 67% C4 32 NRP1- 400 D1 253 238 ± 22 40% B1B2-mFcD2 222 (1 μg/mL) 200 E1 121  122 ± 1.5 39% E2 123 100 F1 70 65 ± 7 36%F2 60 NRP1- 400 D3 230 214 ± 23 46% B1B2-mFc D4 197 (10 μg/mL) 200 E3112  107 ± 7.2 47% E4 102 100 F3 41 41 ± 1 59% F4 42 0.5% BSA 400 A5 318 323 ± 7.4 20% A6 328 200 B5 167  166 ± 1.2 17% B6 165 100 C5 77 73 ± 527% C6 70

Example 2 Selective Depletion Analysis

The differential effect of NRP1 binding on VEGF-A₁₆₅ versus VEGF-A₁₂₁was determined in a plate pull-down assay. Plates were coated with 10μg/mL human NRP-1 in phosphate buffered saline and 0.5% bovine serumalbumin (BSA). Various concentrations of VEGF-A₁₆₅ or VEGF-A₁₂₁ wereincubated in the coated plates for four hours to overnight. The resultsare presented in Table 2. In summary, from about 38% to about 65% of theVEGF-A₁₆₅ was depleted by the hNRP-1; whereas, no more than about 21% ofthe VEGF-A₁₂₁ was depleted. The amount of VEGF-A remaining in thesupernatants post-hNRP1 pull-down was determined via ELISA.

TABLE 2 Starting concentration of VEGF isoform Post-hNRP-1 pull-down(ρg/mL) concentration (ρg/mL) Percent change VEGF-A₁₆₅ 250 90.1 −65% 12549.8 −58% 62.5 25.3 −54% 31.3 14.3 −52% 15.6 8.1 −57% 7.8 6.2 −38% 3.93.4 −60% VEGF-A₁₂₁ 250 196.39 −21% 125 107.17 −15% 62.5 56.39 −10% 31.330.70  −5% 15.6 16.39  4% 7.8 10.18  31% 3.9 6.30  62%

Example 3 VEGF-A₁₆₅ Depletion in Plasma or Serum

The differential effect of NRP1 binding on VEGF-A₁₆₅ and VEGF-A₁₂₁ in aplate pull-down assay was determined. Plates were coated with 10 μg/mLhuman NRP-1 in phosphate buffered saline and 0.5% bovine serum albumin(BSA). Various concentrations of VEGF-A₁₆₅ or VEGF-A₁₂₁ (250, 125, 62.5,31.3, 15.6, 7.8, and 3.9 ρg/mL) were prepared in (a) assay diluent, (b)assay diluent+50% v/v pooled human plasma, (c) assay diluent+10% v/vpooled human plasma, (d) assay diluent+50% v/v pooled human sera, and(e) assay diluent+10% v/v pooled human sera. These VEGF-A solutions wereincubated in the coated plates for four hours to overnight. The averagepercent depletion from all concentrations of VEGF-A isoforms arepresented in Table 3. The amount of VEGF-A remaining in the supernatantspost-hNRP1 was determined via ELISA.

Example 4 Depletion Ratios

Various ratios of VEGF-A₁₆₅ to VEGF-A₁₂₁ were made in assay diluent andsubjected to hNRP1 pull-down, as described in Examples 2 and 3. Theresults are presented in Table 4, which demonstrates the selectivepull-down of the 165 isoform over the 121 isoform of VEGF-A.

TABLE 3 Average percent depletion Average percent depletion ProtocolVEGF-A₁₆₅ VEGF-A₁₂₁ Diluent 67-70% 11-12% 50% plasma 67-70% NA 10%plasma 70-75% 12-13% Diluent 65-70% NA 50% sera 60-65% NA 10% sera70-72% NA

TABLE 4 Percent loss Ratio of VEGF-A_(total) VEGF-A₁₆₅ (225ρg/mL):VEGF-A₁₂₁ (25 ρg/mL) 70% VEGF-A₁₆₅ (125 ρg/mL):VEGF-A₁₂₁ (125ρg/mL) 40% VEGF-A₁₆₅ (25 ρg/mL):VEGF-A₁₂₁ (225 ρg/mL) 12%

What is claimed is:
 1. A method of treating cancer, comprising, (a)obtaining a blood sample from a patient who suffers from cancer; (b)enriching the amount of VEGF-A121 in the sample by the steps of:contacting the sample comprising VEGF-A165 and VEGF-A121 with (1) aneuropilin 1 (“NRP1”) protein, or (2) a fraction of the NRP1 proteincomprising the B1B2 domain, wherein the VEGF-A165 binds to the NRP1protein or fraction thereof to form a NRP1-VEGF-A165 complex; andseparating the sample from the NRP1-VEGF-A165 complex to form aVEGF-A165-depleted sample; (c) quantifying the total remaining VEGF-A inthe VEGF-A165-depleted sample; wherein the total remaining VEGF-A isenriched for VEGF-A121; and (d) administering a VEGF antagonist, if thetotal remaining VEGF-A is above a threshold amount.
 2. The method ofclaim 1, wherein the NRP1 protein or fraction thereof is fixed to asubstrate prior to step (b)(i).
 3. The method of claim 2, wherein thesubstrate is a bead, a plate or a well of a microtiter plate.
 4. Themethod of claim 3, wherein the bead, plate or well is coated with asolution of NRP1 protein.
 5. The method of claim 4, wherein the bead,plate or well is coated with NRP1 protein by applying a 1 μg/mL-10 μg/mLsolution of NRP1 protein to the plate or well.
 6. The method of claim 1,wherein the NRP1 protein is human NRP1.
 7. The method of claim 1,wherein the NRP1 protein is a fusion protein comprising a NRP1 proteindomain comprising a B1B2 domain and an Fc domain.
 8. The method of claim7, wherein the NRP1-fusion protein consists essentially of NRP1 domainsB1 and B2 fused to an Fc domain.
 9. The method of claim 7, wherein theNRP1-fusion protein consists essentially of the extracellular domain ofNRP1 fused to an Fc domain.
 10. The method of claim 1, wherein thesample is additionally contacted with heparin at step (a).
 11. Themethod of claim 10, wherein the heparin is at a concentration of about1-10 μg/mL.
 12. The method of claim 1, wherein the VEGF-A165-depletedsample contains less than 65% of the VEGF-A165 of the non-depletedsample.
 13. The method of claim 1, wherein the VEGF-A165-depleted samplecontains less than 34% of the VEGF-A165 of the non-depleted sample. 14.The method of claim 1, wherein the blood sample comprises plasma orserum.
 15. The method of claim 1, wherein the VEGF-A121-enrichedfraction is quantified via ELISA using an anti-VEGF antibody.
 16. Themethod of claim 1, wherein the VEGF antagonist is selected from thegroup consisting of bevacizumab, ranibizumab, aflibercept, andramucirumab.
 17. The method of claim 1, wherein the cancer is selectedfrom the group consisting of metastatic breast cancer, gastric cancer,pancreatic cancer, renal cell carcinoma, non-small cell lung cancer,androgen-independent prostate cancer, ovarian cancer, adenocarcinoma,and colorectal cancer.